[unreadable] The proposed research seeks to develop selectable markers based on "natural" amino acid auxotrophies and sugar sensitivities exhibited by wild-type Francisella strains. We have identified one such F. novicida auxotrophy, for histidine, which can be satisfied by histidinol if the product of the hisD gene is supplied expressed under a Francisella promoter. Thus, growth on minimal with histidinol in place of histidine provides a selection for the hisD+ marker. We will examine whether it is possible to find an analogous condition in which functions catalyzing the last step of methionine biosynthesis can likewise be utilized for selection. In addition, we will examine whether any of several sugar sensitivities of the wild-type strain such as for xylose can be developed for selection. Our overall plan is to screen for the sensitivities and develop the markers in F. novicida as a surrogate for the highly virulent F. tularensis strain. The final step of the project will be to use one or more of the markers to generate cloning plasmids and transposons which function in multiple Francisella species. (Some of the experiments included in the final step involve select agent strains; all such experiments will be carried out as part of a project supported independently through the WWAMI Research Centers of Excellence.) [unreadable] [unreadable] [unreadable]